A mechanism-based potent sirtuin inhibitor containing Nε-thiocarbamoyl-lysine (TuAcK)

Abstract

In the current study, we have identified N(ε)-thiocarbamoyl-lysine (TuAcK) as a general sirtuin inhibitory warhead which was shown to be able to confer potent sirtuin inhibition. This inhibition was also shown to be mechanism-based in that the TuAck residue was able to be processed by a sirtuin enzyme with the formation of a stalled S-alkylamidate intermediate.

Figure 1

(a) The proposed chemical mechanism for the sirtuin-catalyzed lysine N^ε de(thio)acetylation reaction. When a ThAcK-containing suicide substrate (or mechanism-based inhibitor) (X = S) is used, the corresponding α-1′-S-alkylamidate intermediate is stalled along the reaction coordinate. ADP, adenosine diphosphate; B: refers to a general base. (b) Structural comparison of N^ε-acetyl-lysine (AcK) and its analogs.

Figure 2

Western blot analysis of the p53 protein acetylation level change in HCT116 human colon cancer cells following the treatment with compound 1.

Figure 3

The MALDI-MS analysis of the following samples: (a) The assay mixture from compound 1 with SIRT1 and NAD⁺; (b) The assay mixture from compound 1 with NAD⁺ in the absence of SIRT1; (c) The assay mixture from NAD⁺ with SIRT1 in the absence of compound 1; (d) the MALDI matrix 3-hydroxypicolinic acid (3-HPA) only. All the MALDI-MS analyses were performed in a negative reflector mode. See ref. for further experimental conditions.

Scheme 1

Synthesis of the TuAcK-containing compound 1. Reagents: (i) Fmoc-based SPPS; (ii) 85% (v/v) TFA in DCM; (iii) Thiophosgene (CSCl₂), Et₃N; (iv) 7 N NH₃ in MeOH.